Development Of Bionic Cartilage-matrix Hydrogel Mainly Constituted By TypeⅡ Collagen

Trauma in joint can cause articular disability, which has its symptoms including pain,deformity, low mobility, and so on. Sickness in articular cartilage is very important in jointtrauma. Nutrition of cartilage mainly derives from penetration exchange with synovial fluidbut no blood supply and nerve growing. Once pain is felt, cartilage disease has been in endstage. At the same time, there is little effective clinical therapies for cartilage disease.At present, some clinical strategies are safe and effective for cartilage sickness with themethod of tissue engineering, such as ACI, MACI, etc. The appearances of these comediespredict the trend of cartilage defect repairing by tissue engineering cartilage therapy infuture.However,their application in cartilage sickness is limited, because of its inadequateautologous chondrocytes source,secondary surgery and donor site complication.In this research, typeⅡ collagen were extracted from porcine hyaline cartilage on thebasis of micro-invasive and bionic theory, and its purity and components were identifiedprimarily; typeⅡ collagen hydrogel were prepared,and its physicochemical characters wereinvestigated primarily, such as degeneration rate and swell rate;With mixing seed cells inhydrogel, cell compatibility was evaluated by histological section,meanwhile histologicalcompatibility was evaluated in KM mice in1w,3w,9w by subcutaneous implantation ofhydrogel; Then,CS(chondroitin sulfate),HA(hyaluronic acid), COLⅡ were mixed on theproportion of normal extracellular matrix, and composite CS-HA-COLⅡ hydrogel wereprepared on that basis.Based on the previous study, effectiveness of cartilage defects repairedby typeⅡ collagen and COLⅡ-CS-HA hydrogel were investigated by gross observation,stereotype microscope,3.0T MRI， histological staining and immunohistochemicalstaining,respectively. In4weeks, repairing tissues in full-thickness cartilage defects inporcine knee could be found in COLⅡand COLⅡ-CS-HA group,and content of typeⅡcollagen in repairing tissues was better in COLⅡ-CS-HA group than COLⅡgroup.Methods:1. Extraction and identification of COLⅡ. TypeⅡ collagen were extracted from hyaline cartilage in porcine knee,and itsquality,purity were both evaluated by SDS-PAGE,amino acid analysis,safranin O staining andshaking-bacteria experiment.2. Physicochemical parameters, biological compatibility of typeⅡcollagen hydrogelwere investigated,respectively.①TypeⅡ collagen hydrogel were prepared with base-neutralizing,which had theability of phase-change.②Degradation rate,swell rate,and water content of hydrogel were observed primarilyby hydrolysis experiment and cool-dry experiment in vitro.③COLⅡhydrogel-bone marrow derived stem cells(mainly MSCs) were cultured on4w,and its cell compatibility was evaluated by histological section with HE staining,fastgreen-safranin O staining,alcian blue staining.④Histological compatibility of hydrogel was observed by histological section insubcutaneous implantation of hydrogel in mice at1w,3w,9w.3.COLⅡ-CS-HA composite hydrogel were prepared biomimeticly on the basis ofprevious experiment,and its general properties and microstructure were observed,respectively.4.Phenomenons of repairing effect in full-thickness cartilage defects in porcine kneetreated by COLⅡand COLⅡ-CS-HA hydrogel were observed in short period.①3.0T MRI②general observation and stereoscopy.③Histological staining④Immunohistochemical stainingResults1. Extraction and identification of COLⅡ:the concentration of typeⅡ collagen was66mg/ml, and had similar proteomic characteristics with COLⅡ by SDS-PAGE and aminoacid analysis.2. Preparation and evaluation of COLⅡhydrogel.①Good pore distribution and connection were observed in the hydrogel by SEM.②Hydrolysis experiment showed that hydrolysis rate of the hydrogel was nearly30%;swell rate was2684%;water-containing rate was above90%.③Cell compatibility experiment showed that seeding cells were alive,can proliferate normally without obvious cell swelling and nucleus dissolving.④Subcutaneous implantation experiment showed that hydrogel were absorbedcompletely in9w,without inflammatory cells aggregating.3. Good pore distribution and connection were observed in the hydrogel by SEM.4. Large animal experiment①3.0T MRI:cartilage layer can be monitored clearly,.In hydrogel-cell group,obvious synovial membrane hyperplasia and joint effusion were not found;medial signalscould be found in defect,and obvious abnormal signal were not found in subchondral boneand deep tissue;in hydrogel group(no seeding cells),unclear signal were found in defect andabnormal signal could be found in subchondral bone.②General observation and stereoscope showed that hydrogel-cell group(includingCOL Ⅱ and COLⅡ-CS-HA group) had better filling quality in appearance than the hydrogelgroup without mixing cell.Additionally,white repairing tissue could be found in defects inhydrogel-cell group.③Histological and immunohistochemical staining:differentiated chondrocyte werefound in defects in fast green-safranin O staining and alcian blue staining;positive stainingcould be found in the border and bottom of defects,which implied cartilage proteoglycanexcreted by chondrocytes; content of typeⅡcollagen and density of cells in defect fillingswere higher in COLⅡ-CS-HA-cells hydrogel than in COLⅡ-cells hydrogel,which impliedthat repairing reaction and quality of restoration were better in COLⅡ-CS-HA-cells hydrogelthan in COLⅡ-cells hydrogel.Conclusions:1. Extracted COL Ⅱhad good purity and concentration,and its proteomic property wassimilar with COL Ⅱ.2. The typeⅡ collagen hydrogel had better cell and histological compatibility.3. Composite hydrogel constituted with COL Ⅱ-CS-HA was developedbiomimeticly;good repairing effect could be found in full-thickness cartilage defects in femurtrochlear in porcine knee,and fillings approximate with hyaline cartilage in short-term largeanimal experiment.However,some physicochemical properties(including viscoelasticity,enzymedegradation property, etc) and its biological safety(including acute and chronic inflammation,hemolytic reaction and genotoxcity etc) were not investigated intensively.Theseshortcomings will be overcome in future.